The single most important consumer-defined quality characteristic of fruit is flavor and in most fruit, consumer flavor preferences are dominated by sweetness. There is a well-characterized difference in sweetness of hexose sugars, with fructose being approximately twice as sweet as its hexose isomer, glucose.
The proportion of fructose in fruit is controlled, at least in part, by its conversion to fructose-6-phosphate by the hexose kinase, fructokinase. The hexose kinases can be divided into three general categories according to their hexose substrate specificity. Hexokinase (HK) can phosphorylate glucose and fructose, while glucokinase (GK) and fructokinase (FK) are relatively specific for the respective hexose isomer. Plant tissues may contain multiple hexose kinases and multiple isozymes of the enzymes.
Carbohydrate metabolism and carbohydrate composition has previously been modified in transgenic plants by increasing starch levels or by altering the relative levels of sucrose in fruit. Several studies with fruit tissues have indicated that during the development and ripening of hexose-accumulating fruit, imported sucrose can be hydrolyzed by a hexose invertase into its two hexose moieties; fructose and glucose. The possible fates of the fructose and glucose moieties of imported sucrose after invertase-mediated hydrolysis are illustrated in FIG. 1. According to this model, the balance of the activities of hexokinase (HK), fructokinase (FK) and the hexose phosphatases (These enzymes dephosphorylate hexose-6-phosphates into hexoses) control levels of glucose-6-phosphate (Glc-6-P) and fructose-6-phosphate (Fru-6-P). Glc-6-P in turn isomerizes via phosphoglucose isomerase (PGI) to Fru-6-P, a reversible reaction that is predicted to be approximately at equilibrium in vivo. Fru-6-P then enters respiratory pathways. It is postulated these reactions serve to equilibrate Fru-6-P and Glc-6-P pools in fruit tissues, and as a consequence, also equilibrate glucose and fructose pools.
A fructokinase gene has been previously cloned from potato and the sequence of its cDNA determined (Smith, S. B., et al., Plant Physiol. 102:1043 (1993); Taylor, M. A., et al., J. Plant Physiol. 145:253 (1995)). In addition, fructokinase isozymes have been isolated and at least partially purified from barley leaves (Baysdorfer, C., et al., J. Plant Physiol. 134:156 (1989)); taproots of sugar beets (Chaubron, F., et al., Plant Science 110:181 (1995)); pea seeds (Copeland, 1., et al., Plant Physiol. 62:291 (1978)); maize kernels (Doehlert, D. C., Plant Physiol. 90:353 (1990)); and tomatoes (Martinez-Barajas, E., and Randall, D. D., Planta 199:451 (1996)).
Previous research has used traditional breeding practices to select for fruit varieties with enhanced sweetness. This has been achieved in many different varieties, e.g., super-sweet corn and melons. In all cases, the increase in sweetness has been achieved by selecting for varieties with a higher concentration of total soluble sugars. However, in other fruit, such as the tomato, increases in the concentration of total soluble sugars is associated with a decline in total yield. Modern varieties, which have been selected to be high yielding, tend to have lower total sugar levels and reduced sweetness, relative to older cultivars. Thus, the prior art fails to provide a cost-effective means of producing plants with increased fructose levels but without undesired traits. The present invention addresses these and other needs.